LOCUS       sequence-384004-        7945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Marker for yeast mitochondria. Integration of 2xGFP tag at CIT1 C 
            terminus. Uses auxotrophic marker URA3(Candida albicans). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7945)
  AUTHORS   Li D, Yang SG, He CW, Zhang ZT, Liang Y, Li H, Zhu J, Su X, Gong Q, 
            Xie Z
  TITLE     Excess diacylglycerol at the endoplasmic reticulum disrupts 
            endomembrane homeostasis and autophagy.
  JOURNAL   BMC Biol. 2020 Aug 28;18(1):107. doi: 10.1186/s12915-020-00837-w.
  PUBMED    32859196
REFERENCE   2  (bases 1 to 7945)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Biol.";
            date: "2020-08-28"; volume: "18(1):107. doi"; pages: " 
            10.1186/s12915-020-00837-w"
COMMENT     Sequence Label: CIT1-2EGFP-ter-URA3
FEATURES             Location/Qualifiers
     source          1..7945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1320..2033
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     CDS             2070..2786
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2070..2783
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2807..2994
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2862..2886)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3277..3310
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      4265..4462
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4516..4549
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5589..5608)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5590..5608)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5695..5712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5866..6454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5946..5965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6625..7416,7417..7485))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7248..7267
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7486..7590)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7658..7676
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7714..7736)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7836..7855
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7936..7945,1..9)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7936..7945,1..8)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 cactatagaa cgcggccgcc agctgaagct tcggtaaaac cgttattggt gaagttcttt
       61 tggagcaagc ttatggtggt atgagaggta ttaaaggcct tgtttgggaa ggttccgtgt
      121 tagaccccga agaaggtatt agatttaggg gtcgtactat tccagaaatt caaagggaac
      181 taccaaaggc tgagggtagt acagaacctt tgccagaagc tttattttgg ttgcttttga
      241 ctggtgaaat acctactgac gctcaagtta aagccctttc tgctgattta gctgccagat
      301 cagaaattcc agagcacgtt atccaacttt tagatagcct cccaaaagat ctacatccaa
      361 tggcgcaatt ttctattgcc gtgactgctt tagaaagcga gtctaagttt gccaaagcat
      421 atgctcaagg tgtatccaag aaagaatatt ggagctatac atttgaagat tcgttagatc
      481 tgctgggtaa attacctgtt attgcttcca aaatttatcg taatgtgttc aaggatggta
      541 aaattacttc aaccgatcct aatgctgact atggtaaaaa tttggcccaa cttttgggct
      601 acgaaaacaa ggattttatt gacttaatga gactatattt aactattcat tctgatcatg
      661 aaggtggtaa cgtttctgcc catactacac atttagtggg ttctgcctta tcttcgccat
      721 acttatcttt ggccgctggt ttgaatggtt tagctggccc attacatggt cgtgccaatc
      781 aagaagtttt agaatggcta tttaaattga gagaagaagt gaaaggtgac tattcaaaag
      841 aaacaattga aaagtacttg tgggatactt tgaacgcagg gagagttgtt cctggttatg
      901 gccatgcggt tttgagaaaa actgatcctc gttatacggc tcaacgtgaa ttcgcattga
      961 aacatttccc agattacgag ttatttaagt tggtctccac catttatgaa gttgccccag
     1021 gggttttaac taagcatggt aaaactaaga acccatggcc aaatgttgat tcacattccg
     1081 gtgttttatt gcaatactat ggtctaactg aggcttcgtt ctacactgta ttgtttggtg
     1141 ttgccagagc tattggtgtg ttaccccaat taatcatcga tagggctgtt ggtgctccaa
     1201 tcgaaaggcc aaaatcattc tccaccgaaa aatacaagga gttggtaaag aaaatcgaaa
     1261 gtaagaacgc ttcgtacgct gcaggtcgac ggatcggtga cggtgctggt ttaattaaca
     1321 tgtctaaagg tgaagaatta ttcactggtg ttgtcccaat tttggttgaa ttagatggtg
     1381 atgttaatgg tcacaaattt tctgtctccg gtgaaggtga aggtgatgct acttacggta
     1441 aattgacctt aaaatttatt tgtactactg gtaaattgcc agttccatgg ccaaccttag
     1501 tcactacttt aacttatggt gttcaatgtt tttctagata cccagatcat atgaaacaac
     1561 atgacttttt caagtctgcc atgccagaag gttatgttca agaaagaact atttttttca
     1621 aagatgacgg taactacaag accagagctg aagtcaagtt tgaaggtgat accttagtta
     1681 atagaatcga attaaaaggt attgatttta aagaagatgg taacatttta ggtcacaaat
     1741 tggaatacaa ctataactct cacaatgttt acatcatggc tgacaaacaa aagaatggta
     1801 tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg ttctgttcaa ttagctgacc
     1861 attatcaaca aaatactcca attggtgatg gtccagtctt gttaccagac aaccattact
     1921 tatccactca atctgcctta tccaaagatc caaacgaaaa gagagaccac atggtcttgt
     1981 tagaatttgt tactgctgct ggtattaccc atggtattga tgaattgtac aaaattccag
     2041 ttccaacgcg tgatctagag aagccattca tgtctaaagg tgaagaatta ttcactggtg
     2101 ttgtcccaat tttggttgaa ttagatggtg atgttaatgg tcacaaattt tctgtctccg
     2161 gtgaaggtga aggtgatgct acttacggta aattgacctt aaaatttatt tgtactactg
     2221 gtaaattgcc agttccatgg ccaaccttag tcactacttt aacttatggt gttcaatgtt
     2281 tttctagata cccagatcat atgaaacaac atgacttttt caagtctgcc atgccagaag
     2341 gttatgttca agaaagaact atttttttca aagatgacgg taactacaag accagagctg
     2401 aagtcaagtt tgaaggtgat accttagtta atagaatcga attaaaaggt attgatttta
     2461 aagaagatgg taacatttta ggtcacaaat tggaatacaa ctataactct cacaatgttt
     2521 acatcatggc tgacaaacaa aagaatggta tcaaagttaa cttcaaaatt agacacaaca
     2581 ttgaagatgg ttctgttcaa ttagctgacc attatcaaca aaatactcca attggtgatg
     2641 gtccagtctt gttaccagac aaccattact tatccactca atctgcctta tccaaagatc
     2701 caaacgaaaa gagagaccac atggtcttgt tagaatttgt tactgctgct ggtattaccc
     2761 atggtatgga tgaattatat aaataaggcg cgccacttct aaataagcga atttcttatg
     2821 atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag
     2881 tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag
     2941 gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagatc
     3001 cgctagggat aacagggtaa tatagatctg tttagcttgc ctcgtccccg ccgggtcacc
     3061 cggccagcga catggaggcc cagaataccc tccttgacag tcttgacgtg cgcagctcag
     3121 gggcatgatg tgactgtcgc ccgtacattt agcccataca tccccatgta taatcatttg
     3181 catccataca ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcgga
     3241 cctgcgagca gggaaacgct cccctcacag acgcgtataa cttcgtataa tgtatgctat
     3301 acgaagttat tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa aggttaggat
     3361 ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg atacagttct
     3421 cacatcacat ccgaacataa acaaccatga cagtcaacac taagacctat agtgagagag
     3481 cagaaactca tgcctcacca gtagcacaac gattatttcg attaatggaa ctgaagaaaa
     3541 ccaatttatg tgcatcaatt gatgttgata ccactaagga attccttgaa ttaattgata
     3601 aattgggtcc ttatgtatgc ttaatcaaga ctcatattga tataatcaat gatttttcct
     3661 atgaatccac tattgaacca ttattagaac tttcacgtaa acatcaattt atgatttttg
     3721 aagatagaaa atttgctgat attggtaata ccgtgaagaa acaatatatt ggtggagttt
     3781 ataaaattag tagttgggca gatattacta atgctcatgg tgtcactggg aatggagtag
     3841 ttgaaggatt aaaacaggga gctaaagaaa ccaccaccaa ccaagagcca agagggttat
     3901 tgatgttagc tgaattatca tcagtgggat cattagcata tggagaatat tctcaaaaaa
     3961 ctgttgaaat tgctaaatcc gataaggaat ttgttattgg atttattgcc caacgtgata
     4021 tgggtggaca agaagaagga tttgattggc ttattatgac acctggagtt ggattagatg
     4081 ataaaggtga tggattagga caacaatata gaactgttga tgaagttgtt agcactggaa
     4141 ctgatattat cattgttggt agaggattgt ttggtaaagg aagagatcca gatattgaag
     4201 gtaaaaggta tagagatgct ggttggaatg cttatttgaa aaagactggc caattataat
     4261 cagtactgac aataaaaaga ttcttgtttt caagaacttg tcatttgtat agttttttta
     4321 tattgtagtt gttctatttt aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga
     4381 catcatctgc ccagatgcga agttaagtgc gcagaaagta atatcatgcg tcaatcgtat
     4441 gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa
     4501 aacgagctcg aattcataac ttcgtataat gtatgctata cgaagttata tcgatgatat
     4561 cagatccact agtggcctat gcggccgcgg cagggtatgc gactattcaa acacgtattt
     4621 tcatatacga atgagtagtt tataatttat ataaatatat attacataag gaaatggcta
     4681 tgacttcatg atttttacgc tttgagtccc ctatgcctac cggttatgtt atatacttgg
     4741 caaatgatat caatgaatag aaaacaaaat gctcttaaat tcctgcaaag cgctttcaaa
     4801 tcgaattgtg gtgggctggc gcgctgattt agcagtaaaa tctagcctgt agtcgatata
     4861 ctacgtcaag gtgtaactaa cgtttatatg tatatgaacc tgcagaagaa gcaaaatgac
     4921 aaggttaatg catctttatc tgtcctagag tagaagatac actaaaaagt tgataaagcg
     4981 aatataatat ggtcatgtga aaaaagcaat ctcataaaat tttctctttt tccttttttt
     5041 cttctgttct tacctcaaga taaatgtcac ttttatacta attaaaagca gtgttttata
     5101 tttctaccag caaggttttg agtttaatca cataaaacta ggagcagggt agcataaacc
     5161 tgctgtctaa tccgtattca ttcttatcag tccagcgccg tttggggcac ttttagtaaa
     5221 cagtaactta taccacatta atataatttc ttagttcggc atttttttgc taggcgccta
     5281 aacgcagata tccttaaata ccaaatttga acgttatttt acttttcgga agacaaatac
     5341 ataaataaat tttaacatta atatataaaa aaataataaa aaaatgtata ttttttaaaa
     5401 tgttttaaaa tatttttaaa aagctgcacg tgactaacta gaaccatttc acaacaactc
     5461 aattaattgc ggcttcacat ctacttgctt atagggaaga agtaaagaga ataatccgat
     5521 ttattatagg gttgcaaatt ttatgtgtac attacttttt tcttacatag ccgcggatct
     5581 gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg
     5641 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5701 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5761 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5821 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5881 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5941 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6001 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6061 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6121 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6181 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6241 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6301 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6361 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6421 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6481 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6541 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6601 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6661 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6721 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6781 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6841 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6901 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6961 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7021 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7081 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7141 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7201 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7261 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7321 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7381 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7441 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7501 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7561 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7621 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7681 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     7741 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     7801 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     7861 agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg
     7921 tatcatacac atacgattta ggtga
//
